Methods for rare event detection

ABSTRACT

The invention provides methods for detecting small mutations and structural alterations in DNA by using binding proteins to protect those features while digesting unprotected DNA in a sample. To detect small mutations, a protein that binds exclusively to the mutation of interest, and not to wild-type, is used. For structural alterations, binding proteins are used that flank a breakpoint of the alteration. After digestion of unbound, unprotected nucleic acid in the sample, the mutation- or breakpoint-containing segment remains as an isolated DNA fragment. The sample is then assayed to detect any fragment of DNA and the detection of the fragment indicates the presence of the mutation or breakpoint in the subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of, and priority to, U.S.Provisional Application No. 62/568,121, filed Oct. 4, 2017, U.S.Provisional Application No. 62/526,091, filed Jun. 28, 2017, and U.S.Provisional Application No. 62/519,051, filed Jun. 13, 2017, thecontents of each of which are incorporated by reference.

TECHNICAL FIELD

The disclosure relates to molecular genetics.

BACKGROUND

DNA has the potential to provide clinically actionable information abouta subject. For example, DNA from a tumor can reveal whether a cancerpatient is in remission, or may inform a physician about immunotherapytreatments that may be effective for the patient. Similarly, fetal DNAcan be studied to detect inherited genetic disorders, aneuploidy, orpreeclampsia. However, a consistent challenge in accessing theactionable genetic information lies in existing approaches to sequencingDNA.

Typical DNA sequencing assays include the use of next-generationsequencing (NGS) platforms to capture, amplify, and sequence a subject'sDNA. However, typical NGS platforms face a number of challenges.Detecting rare mutations such as a mutation in circulating tumor DNA ina plasma sample that also contains an abundance of “normal”, somaticDNA, requires successfully amplifying that tumor DNA for sequencing.Detecting structural alterations such as translocations, inversions,copy number variations, loss of heterozygosity, or large insertions ordeletions (indels) is particularly difficult since NGS platforms give alarge number of short reads for which assembly is made difficult by suchstructural alterations.

SUMMARY

The invention provides methods for detecting small mutations andstructural alterations in DNA by using binding proteins to protect thosefeatures while digesting unprotected DNA in a sample. To detect smallmutations, a protein that binds exclusively to the mutation of interest,and not to wild-type, is used. For structural alterations, bindingproteins are used that flank a breakpoint of the alteration. Afterdigestion of unbound, unprotected nucleic acid in the sample, themutation- or breakpoint-containing segment remains as an isolated DNAfragment. The sample is then assayed to detect the fragment and thedetection of the fragment indicates the presence of the mutation orbreakpoint in the subject.

Embodiments of the invention use proteins that are originally encoded bygenes that are associated with clustered regularly interspaced shortpalindromic repeats (CRISPR) in bacterial genomes. Preferred embodimentsuse a CRISPR-associated (Cas) endonuclease. For such embodiments, thebinding protein in a Cas endonuclease complexed with a guide RNA thattargets the Cas endonuclease to a specific sequence. The complexes bindto the specific sequences in the nucleic acid segment by virtue of thetargeting portion of the guide RNAs. When the Cas endonuclease/guide RNAcomplex binds to a nucleic acid segment, the complex protects thatsegment from digestion by exonuclease. When two Cas endonuclease/guideRNA complexes bind to a segment, they protect both ends of the segment,and exonuclease can be used to promiscuously digest un-protected nucleicacid leaving behind an isolated fragment—the segment of DNA between twobound complexes.

Structural alterations are detected using guide RNAs designed tohybridize to targets flanking a boundary of the alteration. Using twosuch guide RNAs, first and second Cas endonucleases will bind to thenucleic acid in positions that flank the breakpoint, thereby definingand protecting the segment of nucleic acid that includes the breakpoint.In the absence of the alteration, the two Cas endonuclease/guide RNAcomplexes will not bind to the same strand, and all of the nucleic acidwill end up digested upon exposure to exonuclease. Small mutations, suchas substitutions or small indels, are detected using an allele-specificguide RNA—a guide RNA that binds the Cas endonuclease exclusively to themutation of interest. An allele-specific guide RNA may be used inconjunction with another guide RNA that binds a Cas endonuclease to thesame nuclei acid, so that the two Cas endonuclease/guide RNA complexesdefine and protect a segment between them, but only do so when the smallmutation is present in the sample. Accordingly, the invention providesmethods for selectively isolating segments of nucleic acid that containclinically relevant mutations.

Protecting a segment of target nucleic acid with two binding proteinswhile promiscuously digesting unprotected nucleic acid may be describedas a negative enrichment for the target. Embodiments of negativeenrichment may be used for the detection of “rare events” where aspecific sequence of interest makes up a very small percentage of thetotal quantity of starting material. Specifically, negative enrichmenttechniques may be used to detect specific mutations in circulating tumorDNA (ctDNA) in the plasma of cancer patients, or specific mutations ofinterest potentially associated with fetal DNA circulating in maternalplasma. In addition negative enrichment analysis can be applied topurified circulating tumor cells (CTCs).

In one embodiment a single or a cocktail of Cas9/gRNA complex(s) arecreated with the gRNA(s) designed specifically to target a region in thegenome known to be associated with a clinically relevant fusion event.The sample of interest is exposed to both Cas9/gRNA complexes orcocktail of complexes and subsequently analyzed by a negative enrichmentassay.

Thus the invention provides methods for the detection of clinicallyactionable information about a subject. Methods of the invention may beused to with tumor DNA to monitor cancer remission, or to informimmunotherapy treatment. Methods may be used with fetal DNA to detect,for example, mutations characteristic of inherited genetic disorders.Methods may be used to detect and describe mutations and/or alterationsin circulating tumor DNA in a blood or plasma sample that also containsan abundance of “normal”, somatic DNA, Methods may be used for directlydetecting structural alterations such as translocations, inversions,copy number variations, loss of heterozygosity, or large indels. Thesubject DNA may include circulating tumor DNA in a patient's blood orplasma, or fetal DNA in maternal blood or plasma.

In certain aspects, the invention provides a method for detecting astructural genomic alteration. The method includes protecting a segmentof nucleic acid in a sample by introducing Cas endonuclease/guide RNAcomplexes that bind to targets that flank a boundary of a genomicalteration, digesting unprotected nucleic acid, and detecting thesegment, thereby confirming the presence of the genomic alteration. Thedigesting step may include exposing the unprotected nucleic acid to oneor more exonucleases. Preferably, the Cas endonuclease/guide RNAcomplexes include guide RNAs with targeting regions complementary totargets that do not appear on the same chromosome in a healthy humangenome.

After digestion, the protected segment of nucleic acid may be detectedor analyzed by any suitable method. For example, the segment may bedetected or analyzed by DNA staining, spectrophotometry, sequencing,fluorescent probe hybridization, fluorescence resonance energy transfer,optical microscopy, electron microscopy, others, or combinationsthereof. The segment may be of any suitable length. Methods of theinvention are useful for isolation of long fragments of DNA, and thedigesting step may include isolating the segment as an intact fragmentof DNA with a length of at least five thousand bases. Short fragmentsmay be isolated in some embodiments, e.g., fragments with about 50 to afew hundred bases in length.

The method may include providing a report describing the presence of thegenomic alteration in a genome of a subject.

In some embodiments, the sample includes plasma from the subject and thesegment is cell-free DNA (cfDNA). The plasma may be maternal plasma andthe segment may be of fetal DNA. In certain embodiments, the sampleincludes plasma from the subject and the segment is circulating tumorDNA (ctDNA). In some embodiments, the sample includes at least onecirculating tumor cell from a tumor and the segment is tumor DNA fromthe tumor cell.

Aspects of the invention provide a method for detecting a mutation. Themethod includes protecting a segment of a nucleic acid in a sample byintroducing first Cas endonuclease/guide RNA complex that binds to amutation in the nucleic acid and a second such complex that also bindsto the same nucleic acid. The first and second Cas endonuclease/guideRNA complexes bind to the nucleic acid to define and protect a segmentof the nucleic acid, and—by virtue of the mutation-specific binding ofat least the first complex—only bind to, and protect, the segment in thepresence of the mutation. The method includes digesting unprotectednucleic acid and detecting the segment, there confirming the presence ofthe mutation. The digesting step may include exposing the unprotectednucleic acid to one or more exonucleases.

In preferred embodiments, the first Cas endonuclease/guide RNA complexincludes a guide RNA with targeting region that binds to the mutationbut that does not bind to other variants at a loci of the mutation. Thedetecting step may include DNA staining, spectrophotometry, sequencing,fluorescent probe hybridization, fluorescence resonance energy transfer,optical microscopy, electron microscopy, others, or combinationsthereof. The digesting step may include isolating the segment as anintact fragment of DNA, which fragment may have any suitable length(e.g., about ten to a few hundred bases, a few hundred to a few thousandbases, at least about five thousand bases, etc.). The method may includeproviding a report describing the presence of the mutation in a genomeof a subject.

In some embodiments, the sample includes plasma from the subject and thesegment is cell-free DNA (cfDNA). For example, the plasma may bematernal plasma and the segment may be of fetal DNA. In certainembodiments, the sample includes plasma from the subject and the segmentis circulating tumor DNA (ctDNA). Optionally, the sample includes atleast one circulating tumor cell from a tumor and the segment comprisestumor DNA from the tumor cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 diagrams a method for detecting a structural genomic alteration.

FIG. 2 illustrates a sample that includes DNA from a subject.

FIG. 3 shows binding proteins protecting a DNA segment that includes abreakpoint.

FIG. 4 shows the detection of an isolated segment of nucleic acid.

FIG. 5 shows a report describing a structural alteration in nucleic acidfrom a subject.

FIG. 6 diagrams a method for detecting a mutation.

FIG. 7 illustrates an allele-specific guide RNA for mutation detection.

FIG. 8 illustrates a negative enrichment.

FIG. 9 shows a kit of the invention.

DETAILED DESCRIPTION

FIG. 1 diagrams a method 101 for detecting a structural genomicalteration. The method 101 includes obtaining a sample that includes DNAfrom a subject. Binding proteins are introduced to protect 113 a segmentof nucleic acid in the sample. The binding proteins bind to specifictargets that flank a boundary of a genomic alteration. The method 101includes digesting 115 unprotected nucleic acid and detecting 125 thesegment, there confirming the presence of the genomic alteration in thesubject. A report 135 may be provided that describes the alteration asbeing present in the subject.

Any suitable structural genomic alteration may be detected using themethod 101. Suitable structural alterations may include, for example,inversions, translocations, copy number variations, or geneduplications. Binding proteins are used that will flank a boundary ofthe structural alteration only when the alteration is present. Forexample, binding proteins may be used that—in the absence of thealteration—bind to different chromosomes of a human genome. Methods ofthe invention are used to detect the alteration in a DNA sample from asubject.

FIG. 2 illustrates a sample 203 that includes DNA 207 from a subject.The DNA 207 may be any suitable DNA and in preferred embodimentsincludes cell-free DNA, such as circulating tumor DNA (ctDNA) or fetalDNA from maternal blood or plasma. The sample may include plasma fromthe subject in which the segment is cell-free DNA (cfDNA). In someembodiments, the sample 203 includes maternal plasma and fetal DNA. Incertain embodiments, ctDNA is in the sample 203. In some embodiments,the sample 203 includes at least one circulating tumor cell from a tumorand the segment comprises tumor DNA from the tumor cell.

Methods may include detection or isolation of circulating tumour cells(CTCs) from a blood sample. Cytometric approaches use immunostainingprofiles to identify CTCs. CTC methods may employ an enrichment step tooptimize the probability of rare cell detection, achievable throughimmune-magnetic separation, centrifugation or filtration. Cytometric CTCtechnology includes the CTC analysis platform sold under the trademarkCELLSEARCH by Veridex LLC (Huntingdon Valley, Pa.). Such systems providesemi-automation and proven reproducibility, reliability, sensitivity,linearity and accuracy. See Krebs, 2010, Circulating tumor cells, TherAdv Med Oncol 2(6):351-365 and Miller, 2010, Significance of circulatingtumor cells detected by the CellSearch system in patients withmetastatic breast colorectal and prostate cancer, J Oncol2010:617421-617421, both incorporated by reference.

In the illustrated example, the DNA 207 has a portion 211 thatoriginated from a first chromosome and a second portion 215 thatoriginated from a different chromosome. By virtue of a translocationbetween the two chromosomes, the DNA 207 includes a breakpoint 219 ofthe translocation. The DNA also includes a first binding target 229 fora first binding protein and a second binding target 225 for secondbinding protein. The two binding targets 229, 225 flank the breakpoint219, which lies in a segment 226 between the two binding targets. Thesample may include other nucleic acid 227 that does not include thetargets or the breakpoint. The method includes binding the bindingproteins to the targets 225, 229.

FIG. 3 shows binding proteins 301 being introduced to protect 113 thesegment 226 of nucleic acid where the breakpoint 219 lies. The bindingproteins 301 bind to specific targets that flank a boundary of a genomicalteration. The depicted method for isolating the segment 226 may bedescribed as a negative enrichment. Contrary to the standard approach ofenriching a specific genomic sequence of interest away from aheterogeneous background of DNA by trying to fish out the sequence ofinterest from the ocean of unwanted sequence, the depicted approachdries up the ocean, leaving behind the target sequence of interest.Methods may be used to perform such an approach to enrich for long DNAfragments (˜50-100 kb) of interest. The fragment may be detected oranalyzed, e.g., sequenced by NGS or a long read sequencing platform suchas Oxford Nanopore or PacBio. Methods may be used to isolate any lengthfragment (e.g., 100 bases, 150 bases, 175 bases, etc. . . . ) thatincludes a boundary of an alteration, such as a breakpoint of a fusionevent.

In a population of DNA where a clinically informative fusion event isnot present, the genomic DNA is digested down to the size of a DNAsequence equivalent to the amount of sequence protected by a singleCas9/gRNA complex. However, in those samples where a clinicallyinformative fusion is present, both binding proteins 301 will be locatedon the same DNA strand and therefore protecting the segment 226 betweenthe proteins 301 from DNA degradation.

In a preferred embodiment, the binding proteins 301 are provided by Casendonuclease/guide RNA complexes. Embodiments of the invention useproteins that are originally encoded by genes that are associated withclustered regularly interspaced short palindromic repeats (CRISPR) inbacterial genomes. Preferred embodiments use a CRISPR-associated (Cas)endonuclease. For such embodiments, the binding protein in a Casendonuclease complexed with a guide RNA that targets the Casendonuclease to a specific sequence. Any suitable Cas endonuclease orhomolog thereof may be used. A Cas endonuclease may be Cas9 (e.g.,spCas9), catalytically inactive Cas (dCas such as dCas9), Cpf1, C2c2,others, modified variants thereof, and similar proteins ormacromolecular complexes. A first Cas endonuclease/guide RNA complexincludes a first Cas endonuclease 303 and a first guide RNA 309. Asecond Cas endonuclease/guide RNA complex includes a second Casendonuclease 304 and a second guide RNA 310.

In the preferred embodiments, the two Cas endonuclease complexes (orsets of complexes if nickases are used) define the locus that includes ajunction of a known chimeric/fusion chromosome/gene, i.e., the boundary219. The complexes protect the segment 226 of nucleic acid that includesthe boundary 219. One or more exonuclease 331 is used to digest 115unprotected nucleic acid. In some embodiments, ExoIII and ExoVII destroyall DNA that does not include both binding/protecting sites. The onlyDNA that remains includes the junction, or boundary 219, of the knownchimera (fusion).

As a result of digestion 115 by exonuclease 331, unprotected nucleicacid 227 is removed from the sample. What remains is the segment 226containing the breakpoint 219, to which the first Cas endonuclease 303and second Cas endonuclease 304 may remain bound. The method 101 furtherincludes detecting 125 the segment 226 as present after the digestionstep. Any suitable detection technique may be used such as, for example,DNA staining; spectrophotometry; sequencing; fluorescent probehybridization; fluorescence resonance energy transfer; opticalmicroscopy; or electron microscopy.

The Cas9/gRNA complexes may be subsequently or previously labeled usingstandard procedures, and single molecule analysis identifyingcoincidence signal of the two Cas9/gRNA complexes located on the sameDNA molecule identifies the presence of the clinically informativefusion of interest. The complexes may be fluorescently labeled, e.g.,with distinct fluorescent labels such that detecting involves detectingboth labels together (e.g., after a dilution into fluid partitions). Thecomplexes may be labeled with a FRET system such that they fluoresceonly when bound to the same segment. Preferred embodiments of analysisdoes not require PCR amplification and therefore significantly reducescost and sequence bias associated with PCR amplification. Sampleanalysis can also be performed by a number of approaches such as NGSetc. However, many analytical platforms may require PCR amplificationprior to analysis. Therefore, preferred embodiments of analysis of thereaction products include single molecule analysis that avoid therequirement of amplification.

Kits and methods of the invention are useful with methods disclosed inU.S. Provisional Patent Application 62/526,091, filed Jun. 28, 2017, forPOLYNUCLEIC ACID MOLECULE ENRICHMENT METHODOLOGIES and U.S. ProvisionalPatent Application 62/519,051, filed Jun. 13, 2017, for POLYNUCLEIC ACIDMOLECULE ENRICHMENT METHODOLOGIES, both incorporated by reference.

FIG. 4 shows the detection 125 of the isolated segment 226 of thenucleic acid. The digestion 115 provides a reaction product 407 thatincludes principally only the segment 226 of nucleic acid, as well asany spent reagents, Cas endonuclease complexes, exonuclease, nucleotidemonophosphates, or pyrophosphate as may be present. The reaction product407 may be provided as an aliquot (e.g., in a micro centrifuge tube suchas that sold under the trademark EPPENDORF by Eppendorf North America(Hauppauge, N.Y.) or glass cuvette). The reaction product 407 may bedisposed on a substrate. For example, the reaction product may bepipetted onto a glass slide and subsequently combed or dried to extendthe fragment 226 across the glass slide. The reaction product mayoptionally be amplified. Optionally, adaptors are ligated to ends of thereaction product, which adaptors may contain primer sites or sequencingadaptors. The presence of the segment 226 in the reaction product 407may then be detected using an instrument 415.

The fragment 226 may be detected, sequenced, or counted. Where aplurality of fragment 226 are present or expected, the fragment may bequantified, e.g., by qPCR.

In certain embodiments, the instrument 415 is a spectrophotometer, andthe detection 125 includes measuring the adsorption of light by thereaction product 407 to detect the presence of the segment 226. Themethod 101 may be performed in fluid partitions, such as in droplets ona microfluidic device, such that each detection step is binary (or“digital”). For example, droplets may pass a light source andphotodetector on a microfluidic chip and light may be used to detect thepresence of a segment of DNA in each droplet (which segment may or maynot be amplified as suited to the particular application circumstance).By the described methods, a sample can be assayed for a genomicstructural alteration using a technique that is inexpensive, quick, andreliable. Methods of the disclosure are conducive to high throughputembodiments, and may be performed, for example, in droplets on amicrofluidic device, to rapidly assay a large number of aliquots from asample for one or any number of genomic structural alterations.

The Cas endonuclease/guide RNA complexes can be designed to flanksuspected gene fusions, or may be designed without a priori knowledge ofany such alteration, but introduced to sample nucleic acid in pairs thatinclude guide RNAs with targeting regions complementary to targets thatdo not appear on the same chromosome in a healthy human genome. Thecomplexes bind to healthy DNA on different chromosomes, so detecting asegment via the described method 101 indicates the presence of astructural alteration in the subject's DNA.

When a genomic structural alteration is thus detected, a report may beprovided 135 to, for example, describe the alteration in a patient.

FIG. 5 shows a report 519 as may be provided in certain embodiments. Thereport preferably includes a description of the structural alteration inthe subject (e.g., a patient). The method 101 for detecting structuralalterations may be used in conjunction with a method of describingmutations (e.g., as described herein). Either or both detection processmay be performed over any number of loci in a patient's genome orpreferably in a patient's tumor DNA. As such, the report 519 may includea description of a plurality of structural alterations, mutations, orboth in the patient's genome or tumor DNA. As such, the report 519 maygive a description of a mutational landscape of a tumor.

Knowledge of a mutational landscape of a tumor may be used to informtreatment decisions, monitor therapy, detect remissions, or combinationsthereof. For example, where the report 519 includes a description of aplurality of mutations, the report 519 may also include an estimate of atumor mutation burden (TMB) for a tumor. It may be found that TMB ispredictive of success of immunotherapy in treating a tumor, and thusmethods described herein may be used for treating a tumor.

Methods of the invention thus may be used to detect and reportclinically actionable information about a patient or a tumor in apatient. For example, the method 101 may be used to provide 135 a reportdescribing the presence of the genomic alteration in a genome of asubject. Additionally, protecting 113 a segment 226 of DNA and digesting115 unprotected DNA provides a method for isolation or enrichment of DNAfragments, i.e., the protected segment. It may be found that thedescribed enrichment technique is well-suited to theisolation/enrichment of arbitrarily long DNA fragments, e.g., thousandsto tens of thousands of bases in length.

Long DNA fragment targeted enrichment, or negative enrichment, createsthe opportunity of applying long read platforms in clinical diagnostics.Negative enrichment may be used to enrich “representative” genomicregions that can allow an investigator to identify “off rate” whenperforming CRISPR Cas9 experimentation, as well as enrich for genomicregions that would be used to determine TMB for immuno-oncologyassociated therapeutic treatments. In such applications, the negativeenrichment technology is utilized to enrich large regions (>50 kb)within the genome of interest.

In preferred embodiments, the invention provides methods 101 fordetecting structural alterations and/or methods for detecting mutationsin DNA.

FIG. 6 diagrams a method 601 for detecting a mutation. The method 601includes obtaining 605 a sample that includes DNA from a subject. Thesample is exposed to a first Cas endonuclease/guide RNA complex thatbinds 613 to a mutation in a sequence-specific fashion. The method 601includes protecting 629 a segment of nucleic acid in a sample byintroducing the first Cas endonuclease/guide RNA complex (that binds toa mutation in the nucleic acid) and a second Cas endonuclease/guide RNAcomplex that also binds to the nucleic acid. Unprotected nucleic acid isdigested 635. For example, one or more exonucleases may be introducedthat promiscuously digest unbound, unprotected nucleic acid. While theexonucleases act, the segment containing the mutation of interest isprotected by the bound complexes and survives the digestion step 635intact. The method 601 includes detecting 639 the segment, thereconfirming the presence of the mutation. A report may be provided 643that describes the mutation as being present in the subject.

The method 601 uses the idea of mutation-specific gene editing, or“allele-specific” gene editing, which may be implemented via complexesthat include a Cas endonuclease and an allele-specific guide RNA.

FIG. 7 illustrates the operation of allele-specific guide RNA formutation detection. A sample 705 may contain a mutant fragment 707 ofDNA, a wild-type fragment 715 of DNA, or both. A locus of interest isidentified where a mutation 721 may be present proximal to, or within, aprotospacer adjacent motif (PAM) 723. When the wild-type fragment 715 ispresent, it may contain a wild-type allele 717 at a homologous locationin the fragment 715, also proximal to, or within, a PAM. A guide RNA 729is introduced to the sample that has a targeting portion 731complementary to the portion of the mutant fragment 707 that includesthe mutation 721. When a Cas endonuclease is introduced, it will form acomplex with the guide RNA 729 and bind to the mutant fragment 707 butnot to the wild-type fragment 715. The first Cas endonuclease/guide RNAcomplex includes a guide RNAs with targeting region that binds to themutation but that does not bind to other variants at a loci of themutation.

The described methodology may be used to target a mutation 721 that isproximal to a PAM 723, or it may be used to target and detect a mutationin a PAM, e.g., a loss-of-PAM or gain-of-PAM mutation. The PAM istypically specific to, or defined by, the Cas endonuclease being used.For example, for Streptococcus pyogenes Cas9, the PAM include NGG, andthe targeted portion includes the 20 bases immediately 5′ to the PAM. Assuch, the targetable portion of the DNA includes any twenty-threeconsecutive bases that terminate in GG or that are mutated to terminatein GG. Such a pattern may be found to be distributed over a genome atsuch frequency that the potentially detectable mutations are abundantenough as to be representative of mutations over the genome at large. Insuch cases, allele-specific negative enrichment may be used to detectmutations in targetable portions of a genome. Moreover, the method 601may be used to determine a number of mutations over the representative,targetable portion of the genome. Since the targetable portion of thegenome is representative of the genome overall, the number of mutationsmay be used to infer a mutational burden for the genome overall. Wherethe sample includes tumor DNA and the mutations are detected in tumorDNA, the method 601 may be used to give a tumor mutation burden.

The method 601 includes the described negative enrichment, in which asegment of nucleic acid in a sample is protected 629 by a first Casendonuclease/guide RNA complex (that binds to a mutation in the nucleicacid) and a second Cas endonuclease/guide RNA complex that also binds tothe nucleic acid.

FIG. 8 illustrates operation of the negative enrichment. The sample 705includes DNA 709 from a subject. The sample 705 is exposed to a firstCas endonuclease/guide RNA complex 715 that binds to a mutant fragment707 mutation in a sequence-specific fashion. Specifically, the complex715 binds to the mutation 721 in a sequence-specific manner. A segmentof the nucleic acid 709, i.e., the mutant fragment 707, is protected byintroducing the first Cas endonuclease/guide RNA complex 715 (that bindsto a mutation in the nucleic acid) and a second Cas endonuclease/guideRNA complex 716 that also binds to the nucleic acid. Unprotected nucleicacid 741 is digested. For example, one or more exonucleases 739 may beintroduced that promiscuously digest unbound, unprotected nucleic acid741. While the exonucleases 739 act, the segment containing the mutationof interest, the mutant fragment 707, is protected by the boundcomplexes 715, 716 and survives the digestion step intact.

The described steps including the digestion by the exonuclease 739leaves a reaction product that includes principally only the mutantsegment 707 of nucleic acid, as well as any spent reagents, Casendonuclease complexes, exonuclease 739, nucleotide monophosphates, andpyrophosphate as may be present. The method 601 includes detecting 639the segment 707 (which includes the mutation 721). Any suitabletechnique may be used to detect 639 the segment 707. For example,detection may be performed using DNA staining, spectrophotometry,sequencing, fluorescent probe hybridization, fluorescence resonanceenergy transfer, optical microscopy, electron microscopy, others, orcombinations thereof. Detecting the mutant segment 707 indicates thepresence of the mutation in the subject (i.e., a patient), and the areport may be provided describing the mutation in the patient.

A feature of the method 601 is that a specific mutation may be detectedby a technique that includes detecting only the presence or absence of afragment of DNA, and it need not be necessary to sequence DNA from asubject to describe mutations. The method 601, the method 101, or bothmay be performed in fluid partitions, such as in droplets on amicrofluidic device, such that each detection step is binary (or“digital”). For example, droplets may pass a light source andphotodetector on a microfluidic chip and light may be used to detect thepresence of a segment of DNA in each droplet (which segment may or maynot be amplified as suited to the particular application circumstance).

The method 601 uses a double-protection to select one or both ends ofDNA segments. The gRNA selects for a known mutation on one end. If itdoesn't find the mutation, no protection is provided and the moleculegets digested. The remaining molecules are either counted or sequenced.The method 601 is well suited for the analysis of small portions of DNA,degraded samples, samples in which the target of interest is extremelyrare, and particularly for the analysis of maternal serum (e.g., forfetal DNA) or a liquid biopsy (e.g., for ctDNA).

The method 601 and the method 101 include a negative enrichment stepthat leaves the target loci of interest intact and isolated as a segmentof DNA. The methods are useful for the isolation of intact DNA fragmentsof any arbitrary length and may preferably be used in some embodimentsto isolate (or enrich for) arbitrarily long fragments of DNA, e.g.,tens, hundreds, thousands, or tens of thousands of bases in length orlonger. Long, isolated, intact fragments of DNA may be analyzed by anysuitable method such as simple detection (e.g., via staining withethidium bromide) or by single-molecule sequencing. Embodiments of theinvention provide kits that may be used in performing methods describedherein.

FIG. 9 shows a kit 901 of the invention. The kit 901 may includereagents 903 for performing the steps described herein. For example, thereagents 903 may include one or more of a Cas endonuclease 909, a guideRNA 927, and exonuclease 936. The kit 901 may also include instructions919 or other materials such as pre-formatted report shells that receiveinformation from the methods to provide a report (e.g., by uploadingfrom a computer in a clinical services lab to a server to be accessed bya geneticist in a clinic to use in patient counseling). The reagents903, instructions 919, and any other useful materials may be packaged ina suitable container 935. Kits of the invention may be made to order.For example, an investigator may use, e.g., an online tool to designguide RNA and reagents for the performance of methods 101, 601. Theguide RNAs 927 may be synthesized using a suitable synthesis instrument.The synthesis instrument may be used to synthesize oligonucleotides suchas gRNAs or single-guide RNAs (sgRNAs). Any suitable instrument orchemistry may be used to synthesize a gRNA. In some embodiments, thesynthesis instrument is the MerMade 4 DNA/RNA synthesizer fromBioautomation (Irving, Tex.). Such an instrument can synthesize up to 12different oligonucleotides simultaneously using either 50, 200, or 1,000nanomole prepacked columns. The synthesis instrument can prepare a largenumber of guide RNAs 927 per run. These molecules (e.g., oligos) can bemade using individual prepacked columns (e.g., arrayed in groups of 96)or well-plates. The resultant reagents 903 (e.g., guide RNAs 917,endonuclease(s) 909, exonucleases 936) can be packaged in a container935 for shipping as a kit.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes which come within the meaning andrange of equivalency of the claims are therefore intended to be embracedtherein.

What is claimed is:
 1. A method for detecting a structural genomicalteration, the method comprising: protecting a segment of nucleic acidin a sample by introducing Cas endonuclease/guide RNA complexes thatbind to targets that flank a boundary of a genomic alteration; digestingunprotected nucleic acid; and detecting the segment, thereby confirmingthe presence of the genomic alteration.
 2. The method of claim 1,wherein the digesting step comprises exposing the unprotected nucleicacid to one or more exonucleases.
 3. The method of claim 1, wherein theCas endonuclease/guide RNA complexes include guide RNAs with targetingregions complementary to targets that do not appear on the samechromosome in a healthy human genome.
 4. The method of claim 1, whereinone or more of the Cas endonuclease/guide RNA complexes includes areporter.
 5. The method of claim 5, wherein the reporter includes afluorescent reporter linked to a Cas endonuclease.
 6. The method ofclaim 1, wherein the detecting step includes one selected from the groupconsisting of DNA staining; spectrophotometry; sequencing; fluorescentprobe hybridization; fluorescence resonance energy transfer; opticalmicroscopy; and electron microscopy.
 7. The method of claim 1, whereinthe digesting step comprises isolating the segment as an intact fragmentof DNA with a length of at least five thousand bases.
 8. The method ofclaim 1, further comprising providing a report describing the presenceof the genomic alteration in a genome of a subject.
 9. The method ofclaim 1, wherein the sample includes plasma from the subject and thesegment is cell-free DNA (cfDNA).
 10. The method of claim 9, wherein theplasma is maternal plasma and the segment is of fetal DNA.
 11. Themethod of claim 1, wherein the sample comprises a liquid biopsy.
 12. Themethod of claim 1, wherein the sample includes plasma from the subjectand the segment is circulating tumor DNA (ctDNA).
 13. The method ofclaim 1, wherein the sample includes at least one circulating tumor cellfrom a tumor and the segment comprises tumor DNA from the tumor cell.14. A method for detecting a mutation, the method comprising: protectinga segment of a nucleic acid in a sample by introducing first Casendonuclease/guide RNA complex that binds to a mutation in the nucleicacid and a second Cas endonuclease/guide RNA complex that binds to thenucleic acid digesting unprotected nucleic acid; and detecting thesegment, thereby confirming the presence of the mutation.
 15. The methodof claim 14, wherein the digesting step comprises exposing theunprotected nucleic acid to one or more exonucleases.
 16. The method ofclaim 14, wherein the first Cas endonuclease/guide RNA complex includesa guide RNA with targeting region that binds to the mutation but thatdoes not bind to other variants at a loci of the mutation.
 17. Themethod of claim 14, wherein the detecting step includes one selectedfrom the group consisting of DNA staining; spectrophotometry;sequencing; fluorescent probe hybridization; fluorescence resonanceenergy transfer; optical microscopy; and electron microscopy.
 18. Themethod of claim 14, wherein one or more of the Cas endonuclease/guideRNA complexes includes a reporter.
 19. The method of claim 14, whereinthe digesting step comprises isolating the segment as an intact fragmentof DNA with a length of at least five thousand bases.
 20. The method ofclaim 14, further comprising providing a report describing the presenceof the mutation in a genome of a subject.
 21. The method of claim 14,wherein the sample includes plasma from the subject and the segment iscell-free DNA (cfDNA).
 22. The method of claim 21, wherein the plasma ismaternal plasma and the segment is of fetal DNA.
 23. The method of claim14, wherein the sample includes plasma from the subject and the segmentis circulating tumor DNA (ctDNA).
 24. The method of claim 14, whereinthe sample comprises a liquid biopsy.
 25. The method of claim 14,wherein the sample includes at least one circulating tumor cell from atumor and the segment comprises tumor DNA from the tumor cell.